Researchers at a DOE Bioenergy Research Center use microbes to synthesize a drop-in replacement for standard diesel transportation fuel.

From left, Pamela Peralta-Yahya, Taek Soon Lee, and Mario Ouellet of the DOE Joint BioEnergy Institute team that used microbes to synthesize a bio-based drop-in substitute for diesel fuel.

Today's advanced research on biofuels aims at overcoming two core challenges. First, we want to produce fuels from nonfood plant fiber. Second, we want to produce viable substitutes for gasoline, diesel, and aviation fuel. One key to eventual widespread adoption of next-generation biofuels will be the ability to produce actual drop-in biofuel substitutes for today's petroleum-based transportation fuels. While microbes readily produce ethanol (as every beer and wine drinker knows), it is much more challenging to coax microbes into producing more complicated, high-energy-content molecules with properties similar to those of gasoline or diesel.

Now researchers at a DOE Bioenergy Research Center have used synthetic biology to engineer microbes to produce a bio-based substitute for D2 diesel. (D2 diesel, or #2 diesel, is the form of diesel you find at the gas station pump, used by virtually all diesel-run vehicles around the world.) The compound (which goes by the somewhat complicated name of "bisabolane") closely resembles D2 diesel in both structure and properties. The achievement, by a team of researchers at the DOE Joint BioEnergy Institute (JBEI) at Lawrence Berkeley National Laboratory, may be an important early step toward eventual microbial-based commercial production of drop-in substitutes for petroleum-based fuels.

Zeroing in on a Molecular Target

The research was led by JBEI biochemist Taek Soon Lee, Director of Metabolic Engineering in JBEI's Fuel Synthesis Research Division, and Pamela Peralta-Yahya, a postdoctoral researcher at JBEI. The research was reported in the journal Nature Communications. Co-authors of the article also included Mario Ouellet, Rossana Chan, Aindrila Mukhopadhyay, and Jay Keasling.

JBEI is one of three DOE Bioenergy Research Centers established by the DOE Office of Science in 2007 to accelerate research toward next-generation biofuels. (The other two centers are led, respectively, by Oak Ridge National Laboratory and the University of Wisconsin-Madison in partnership with Michigan State University.)

When Lee and colleagues started this work, they did not know whether the compound in question would be useful as a biofuel, but they liked it based on its chemical structure. The chemical is classified as a terpene—compounds which are found in a wide variety of plants, especially conifers like pine and fir trees, and valued for their fragrance and flavors, such as the smell of green apples, the flavor of hops in beer, or the essential oils found in perfumes. They are a major component of turpentine. Terpenes can also be found in traditional medicine, such as Vitamin A and the anti-malarial drug artemisinin, and are the building blocks of steroids. Plants benefit from having terpenes because the smells that some terpenes emit can attract insects assisting in pollination or act as a deterrent inhibiting animals or microorganisms from eating the plant.

Terpenes, like diesel fuel, are hydrocarbons, meaning that they are made up of carbon atoms linked together to form a chain, much like a string of pearls, with hydrogen molecules attached. Along with the JBEI team, other researchers have started looking at terpenes as a potential biosynthetic diesel, especially so-called sesquiterpenes, or 15-carbon molecules like the JBEI compound, which has a similar size to the average 16-carbon diesel chain (diesel has a range of 10-24 carbon chains).

"Sesquiterpenes have high-energy content and physicochemical properties similar to diesel and jet fuels," Lee said.

Diesel fuel is not just a linear hydrocarbon chain but a mixture of linear, branched, and ringed chains. The differences between these structures can be best described by thinking of roads. Linear chains are long straight roads with no side access on or off; branched chains have streets on the left or right feeding into the main road. A cyclic carbon ring is like a traffic circle in the road. The chemical structure of the JBEI compound has both a branched chain and a single carbon ring. The branching found in the JBEI compound may allow the fuel to tolerate colder temperatures for storage and use, as when an airplane is flying at high altitudes, where temperatures can be around -31°F. Also, the ring structure should increase the fuel density, or the amount of energy per gallon of fuel. Overall the JBEI compound is energy-rich and looks chemically similar to diesel fuel. Now the question became whether it could be converted to work as a usable fuel.

To determine whether the JBEI compound would work as a substitute for diesel in current engines, the researchers needed to test its fuel properties. Beyond its chemical structure, a diesel alternative should have a similar cetane number. The cetane number indicates the combustion quality of the diesel fuel during compression ignition. For the pilot or driver of an aircraft or vehicle, a high cetane number means an engine performs better, runs smoother, has more power, and releases fewer harmful emissions. All the tests showed similar results to D2 diesel, suggesting that the JBEI compound could be a biosynthetic alternative to diesel fuel.

Schematic showing chemical pathway for synthesis of bisabolane from glucose.

Re-Engineering the Microbes

The next big question was how to make large quantities of the JBEI compound cost-effectively. Lee and his group believed the answer lay in using microorganisms.

The researchers believe that although plants are the natural source of terpene compounds, engineered microbial platforms would be the most convenient and cost-effective approach for large-scale production of advanced biofuels. The benefit of using microorganisms over using plants to produce the JBEI compound is that plants require lots of things to grow, including land, water, and fertilizer. Plus, environmental conditions could affect how much of the product is produced. Microorganisms still need water and nutrients, but they can be grown in contained production systems that will not compete with land use for food crops and enable tight control of the growth environment. This also allows them to be "harvested" year round without a concern for the weather or potential pests. Because terpenes can be volatile, meaning that the chemical is released into the air, a contained system could collect them, whereas a plant in a field would release the terpenes into the air.

Lee's group at JBEI engineered what is known as the mevalonate pathway in the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae. Metabolic pathways, like this one, work much like the builders of a house, with one enzyme providing the foundation, another putting up the wall studs, while yet another puts on the roof. The mevalonate pathway creates the simplest single-level house, so that by adding terpene synthases (i.e. bringing in new contractors—in this case enzymes that synthesize various terpenes) you can build on to the basic structure to get a variety of different terpenes, or by following the analogy, houses of different levels and sizes, from two-story houses to six-bedroom mansions.

The method builds on earlier work pioneered, years before the establishment of JBEI, by Jay Keasling, Berkeley Lab Associate Director, University of California Professor, and JBEI CEO, in re-engineering microbes to produce the anti-malarial drug artemisinin.

In the current work, with sugar as the feedstock, Peralta-Yahya and colleagues used the mevalonate pathway to create bisabolene, a precursor chemical or immediate step before generating the JBEI compound, bisabolane. The big question was finding the right synthase. Because terpenes are naturally found in plants, the researchers looked for plants that synthesized the precursor chemical. They found six potential synthase candidates and after testing all the contenders in E. coli and yeast containing the mevalonate pathway, determined that the bisabolene synthase from Abies grandis, a fir tree native to the Pacific Northwest and California, produced the highest amount of bisabolene in the microorganisms.

The final step was to then to isolate the precursor chemical away from the microorganisms and produce the JBEI compound through a final chemical step termed hydrogenation. Hydrogenation reduces or saturates chemical bonds by adding hydrogen and can play an important role in the "state" of a substance. An example of this is the difference between saturated fats (i.e., solid fats, like margarine or shortening) versus unsaturated fats (i.e., liquid fats, like olive oil).

"This is the first report of bisabolane as a biosynthetic alternative to D2 diesel, and the first microbial overproduction of bisabolene in E. coli and S. cerevisiae," said Peralta-Yahya. "This work is a proof-of-principle for advanced biofuels research in that we've shown that we can design a biofuel target, evaluate this fuel target, and produce the fuel with microbes that we've engineered."

Another promising feature is that unlike other biofuels such as ethanol and isobutanol, the JBEI compound was found to be relatively non-toxic to production microbes and could potentially be produced at higher yields.

Lee and colleagues are now preparing to make gallons of the JBEI compound to test in actual diesel engines by using the new Berkeley Lab Advanced Biofuels Process Demonstration Unit (ABPDU) to produce the compound in quantity. The ABPDU is a 15,000 square-foot facility, located near JBEI in Emeryville, California, designed to provide test beds to scale up laboratory discoveries and advance the commercialization of next-generation biofuels. The facility is supported by DOE's Office of Energy Efficiency and Renewable Energy. The ABPDU will also enable the researchers to analyze the production economics and determine the actual cost per gallon of the JBEI compared with diesel.

There are many challenging hurdles ahead before you see next-generation biofuels at your local gas station, including the challenge of developing means of effectively deconstructing plant fiber into sugars that microbes can process—another major research thrust at JBEI and the other two DOE Bioenergy Research Centers. But the ability to use microbes to produce drop-in biofuel substitutes for today's petroleum-based fuels is an important step along the path.